Pulmonary arterial hypertension (PAH) is a severe disease characterized by excessive proliferation of apoptosis-resistant pulmonary artery endothelial cells (PAEC) and smooth muscle cells (PASMC), progressive pulmonary arterial (PA) stiffening, and ultimately right heart failure and death. Recent studies suggest that increased PA stiffness contributes significantly to increased right ventricular after-load and is associated with increased mortality in PAH patients, however the role of PA stiffening in the pathogenesis of PAH has not yet been fully elucidated. We have used atomic force microscopy (AFM) micro-indentation to mechanically characterize the stiffness of pulmonary arteries at an unprecedented micro-scale level in experimental PAH. Our preliminary findings demonstrate that distal pulmonary arteries develop significant increases in matrix stiffness by more than three-fold in the rat models of SU5416/hypoxia and monocrotaline (MCT)-induced PAH. Furthermore, human PASMC and PAEC grown on polyacrylamide substrates with the stiffness of remodeled pulmonary arteries develop a striking hyper-proliferative phenotype, decreased expression of cyclooxygenase (COX)-2, reduced prostaglandin I2 synthesis, and increased secretion of endothelin-1. Taken together, our findings suggest that matrix remodeling in the PA wall fundamentally biases cellular behavior towards progressive vascular remodeling via previously unrecognized effects of matrix stiffening. We hypothesize that increases in PA stiffness are not merely a consequence of pathological alterations in the vessel wall, but rather that increases in matrix stiffness trigger a remodeling phenotype characterized by enhanced cellular proliferation and matrix deposition in pulmonary arteries, promoting mechano-biological feedback amplification of vascular remodeling. To test our hypothesis, we propose three specific aims. In Specific Aim 1, we will investigate the temporal and spatial increases in PA stiffness and reversibility of mechanical changes during experimental PAH. We will utilize AFM micro-indentation to characterize the local mechanical environment of distal pulmonary arteries at the micron spatial scale in the rat models of SU5416/hypoxia and MCT-induced PAH. In Specific Aim 2, we will determine whether increases in matrix stiffness trigger a remodeling phenotype in human PASMC and PAEC and investigate the role of COX-2 in orchestrating these stiffness- dependent cellular alterations. We will investigate the molecular mechanisms by which stiffness modulates COX-2 expression and test whether stiffness-dependent regulation of COX-2-derived prostanoids drives feedback amplification of vascular remodeling. In Specific Aim 3, we will elucidate how stiffness modulates gene expression and identify key transcription factors involved in stiffness-dependent gene regulation in human PASMC and PAEC. We will use transcriptional profiling and bioinformatic approaches, along with a novel dynamic stiffening hydrogel system, to perform an unbiased analysis of temporal gene expression during the stiffness-driven emergence of the hyper-proliferative cellular phenotype.